The principles of differential refractive index (RI) detection are generally well-known. Differential refractive detectors examine the difference in refractive index of a fluid stream (the “sample” path containing one or more analytes to be detected contained within a mobile phase) with that of an analyte-free, often static, fluid sample, namely the “reference.” Various optical methods such as beam deflection, Fresnel reflection or interferometry have been employed to accomplish this measurement. Refractive index detectors are frequently employed for analysis in liquid chromatography, particularly when the analyte(s) of interest lack a suitable UV chromophore.
The refractive index of analyte-bearing solutions is substantially linearly dependent upon analyte concentration. The index difference between the sample and reference fluids often spans a concentration range of nearly 107. Corresponding refractive index differences ranges from 10−9 to 10−2 RI units. For example, water has a refractive index in the visible region of the spectrum of approximately 1.333; a high-end analyte concentration could increase this value to 1.3335 the difference equaling 0.0005 RI units (or 0.5 milliRI units where 1 milliRI=0.001 RI units). Depending upon the analyte and mobile phase the index difference may be positive or negative. A frequently employed optical method is the so-called beam deflection method whereby an optical beam (“light ray”) is directed through a flow cell containing side-by-side prismatic-shaped chambers: the sample fluid flows along the long axis of the chamber—that is—in a direction normal to a plane containing the prismatic or triangular profile. One chamber contains the sample under test while the second contains the reference fluid. The light rays (optical beams) passing through each chamber are refracted to a degree which depends upon the absolute index within each chamber and, upon emerging from the cell will have an overall deflection angle which is relatable to the index difference between the fluids within the chambers. This deflection angle leads to a change in the position of the optical beam at a plane of detection at which is located light detection means such as one or more photodiodes. Thus, in the case of two photodiodes, when there is no refractive index difference each detector reports a substantially equal signal level associated with this balanced condition. If an analyte alters the refractive index of the sample (prism) chamber, one of the detector signals increases while the other decreases since the beam has now been deflected from its initial position. These two signals are processed in such a way as to yield a value for the refractive index difference and, through a calibration step which would precede this experiment, output a reading for the analyte concentration.
A limitation of the conventional prismatic (often a right triangle) shaped chamber is the dispersive character of its geometry. Unlike a circular fluid conduit, the fluid velocity profiles in a triangular shaped conduit are very asymmetric which tends to increase the dispersion or peak width of a chromatographic peak which has eluted from the column. Despite these limitations, a differential RI detector is viewed as an important tool in the arsenal of the chromatographer since it enables the detection of analytes which lack a suitable chromophore for detection by typically more sensitive methods such as UV absorbance or fluorescence. While these limitations are manageable when the incoming chromatographic peak itself is relatively broad, such as might be produced from chromatographic columns with internal diameters in the range of 4.8-7.6 mm and larger, the trend towards smaller column diameters for greater separation efficiencies and reduced solvent consumption means greater attention must be focused on minimizing post-column dispersion, including the contribution from the flow cell itself.